Method for preparing a bone protein preparation and a bone protein preparation
The present invention provides a method for preparing a bone protein preparation which contains for example growth factors. The present invention also provides a bone protein preparation obtained by the method and paste, putty, pellet, disc, block, granule, osteogenic device or pharmaceutical composition containing said bone protein preparation.
1. A method of making a reindeer bone protein guanidine hydrochloride extract comprising
a) demineralizing a reindeer bone in HCl while mixing until the pH remains constant between 2.8 and 3.0 for at least two hours;
b) washing the demineralized reindeer bone of (a) in water, wherein the pH of water in the last washing step is between 2.4 and 2.6; and
c) extracting the washed demineralized reindeer one of (b) with guanidine hydrochloride at a pH of 3.9 to 4.5, wherein said reindeer bone protein guanidine hydrochloride extract comprises Matrix Gla protein, a species of SPP-24 (secreted phosphoprotein), which migrates on SDS-PAGE at approximately 15 kDa, BMP-2, BMP-7 and TGF-beta 1.
2. The method of claim 1 , further comprising filtering the extract of (c) with a microfilter with a cut-off size in the range of 0.1-10 μm to obtain a flow-through fraction of bone protein extract.
3. The method of claim 2 , further comprising filtering the flow-through fraction of bone protein extract with a cassette ultrafilter having a cut-off size in the range of 5-10 kDa to obtain a retentate fraction of bone protein extract.
4. The method of claim 3 , further comprising mixing the retentate fraction of bone protein extract with a polyethylene glycol/glycerol (PEG-GLY) matrix, and combining this mixture with calcium salt granules to generate a bone preparation.
5. The method of claim 3 , further comprising dialyzing the retentate fraction of bone protein extract with water to obtain a wet extract.
6. The method of claim 5 , further comprising re-dissolving the wet extract into guanidine hydrochloride and subsequently dialyzing the re-dissolved guanidine hydrochloride extract with citrate buffer to obtain a precipitate.
7. The method of claim 6 , further comprising mixing the precipitate with a polyethylene glycol/glycerol (PEG-GLY) matrix, and combining this mixture with calcium salt granules to generate a bone preparation.
8. The method of claim 1 , wherein said reindeer bone protein guanidine hydrochloride extract further comprises a species of biglycan, which migrates at approximately 15 kDa on SDS-PAGE.
9. The method of claim 1 , wherein said reindeer bone protein guanidine hydrochloride extract further comprises a species of chondroadherin, which migrates at approximately 15 kDa on SDS-PAGE.
10. The method of claim 8 , wherein said reindeer bone protein guanidine hydrochloride extract further comprises a species of chondroadherin, which migrates at approximately 15 kDa on SDS-PAGE.